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380 Review TRENDS in Science Vol.8 No.8 August 2003

Agrobacterium tumefaciens as an agent of disease

Matthew A. Escobar1 and Abhaya M. Dandekar2

1Department of and , Lund University, So¨ lvegatan 35, Lund, SE-22362, Sweden 2Department of Pomology, University of California, 1 Shields Ave, Davis, CA 95616, USA

Twenty-six years ago it was found that the common lesions on grape roots [4]. A. vitis can survive in planta in soil bacterium tumefaciens is capable of the intercellular spaces of grape tissue without causing extraordinary feats of interkingdom genetic transfer. disease but will initiate tumorigenesis upon tissue Since this discovery, A. tumefaciens has served as a wounding (most commonly frost injury) [4]. Erwin Smith model system for the study of type IV bacterial and C.O. Townsend [5] reported 10 years after the secretory systems, horizontal transfer and bac- discovery of A. vitis that Bacterium tumefaciens (now terial–plant signal exchange. It has also been modified Agrobacterium tumefaciens) was the causal agent of crown for controlled genetic transformation of , a core gall disease in Paris daisy. This organism is capable of technology of plant molecular biology. These areas inducing tumors at wound sites on the stems, crowns and have often overshadowed its role as a serious, wide- roots of hundreds of dicots, although root necrosis is not spread phytopathogen – the primary driver of the first characteristic of A. tumefaciens-mediated crown gall 80 years of Agrobacterium research. Now, the diverse disease [6]. The pioneering work of Cavara and Smith areas of A. tumefaciens research are again converging and Townsend ushered in a century of study of Agrobac- because new discoveries in transformation biology and terium as an agent of disease, a model system of horizontal the use of A. tumefaciens vectors are allowing the gene transfer and a tool for plant transformation (Table 1). development of novel, effective -based Although crown gall disease is not generally fatal strategies for the control of crown gall disease. unless infection occurs in young plants, crown gall related reduction in crop yield and/or vigor can be significant in Members of the Agrobacterium are ubiquitous many perennial horticultural crops [7], such as grape [8], components of the soil microflora, the vast majority of apple [9] and cherry [10]. The decreased productivity of which are saprophytic, surviving primarily on decaying galled plants is probably caused by several factors, organic matter. However, several of agrobacteria including decreased water and nutrient flow owing to cause neoplastic diseases in plants, including Agrobacter- damaged or constricted vasculature at the site of gall ium rhizogenes (hairy root disease), Agrobacterium rubi development, and significant water and nutrient allo- (cane gall disease), Agrobacterium tumefaciens (crown gall cation to the rapidly dividing but unproductive gall sink disease) and Agrobacterium vitis (crown gall of grape). [11–13]. In addition, crown galls are sites for secondary During the past 26 years, it has become apparent that infection by other phytopathogens (e.g. Agrobacterium pathogenesis is a unique and highly syringae and Armillarea mellea) or pests (insect borers), specialized process involving bacterium–plant interking- and can increase plant susceptibility to abiotic stresses dom gene transfer [1]. Crown gall and hairy root have been [6,8,12]. Finally, in planta populations of tumorigenic described as a form of ‘genetic colonization’ [2] in which the agrobacteria can negatively affect graft take, because transfer and expression of a suite of Agrobacterium tumor tissue developing at the graft union prevents fusion in a plant cell causes uncontrolled cell proliferation and of stock and scion tissues [8]. the synthesis of nutritive compounds that can be metab- olized specifically by the infecting . Thus, infection Disease process – transformation and tumorigenesis effectively creates a new niche specifically suited to Agrobacterium pathogenesis requires two basic elements: Agrobacterium survival. This article focuses specifically (1) delivery of tumorigenic DNA into the plant on crown gall, the most agriculturally significant disease (transformation); and (2) the resultant alteration of plant caused by agrobacteria, and the state of current and future cell metabolism, resulting in cell proliferation and the strategies for crown gall disease control. synthesis of nutritive compounds that provide a selective Fridiano Cavara first identified a flagellate, bacilloid advantage for Agrobacterium (tumorigenesis). The focus bacterium (termed Bacillus ampelopsorae) as the causal hereisentirelyonthegall-formingagrobacteria;seeRef.[14] agent of crown gall of grape in 1897 [3]. This organism, for a review of A. rhizogenes and hairy root disease. now called A. vitis, causes the growth of neoplastic tumors on the stem and crown of grapevines and induces necrotic From to integration A detailed treatment of Agrobacterium transformation is Corresponding author: Abhaya M. Dandekar ([email protected]). beyond the scope of this article but several reviews from http://plants.trends.com 1360-1385/03/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S1360-1385(03)00162-6 Review TRENDS in Plant Science Vol.8 No.8 August 2003 381

Table 1. Selected discoveries and insights in Agrobacterium biology

Year Discovery Refs 1853 First written report of crown gall disease. [72] 1897 Agrobacterium vitis identified as the causal agent of crown gall in grape. [3] 1907 Agrobacterium tumefaciens identified as causal agent of crown gall in Paris daisy (Argyranthemum frutescens). [5] 1947 Sterile plant tumor tissue can proliferate indefinitely on hormone-free medium in culture. Tumor cells are [38] proposed to be ‘transformed’ by an Agrobacterium-derived tumor-inducing principle (TIP). 1956 Unusual low molecular weight nitrogenous compounds () are identified exclusively in tumor tissue. [73] 1971 Agrobacterium tumefaciens loses virulence when grown at 378C. The TIP can be transferred between virulent [74,75] and avirulent A. tumefaciens strains. 1974 Agrobacterium tumefaciens virulence depends on the presence of a large ‘tumor-inducing’ (Ti) pasmid. The TIP [76] is probably a component of the Ti . 1977 The T-DNA region of the is present in the genome of crown gall tumor cells: the T-DNA is the TIP. [1] 1980 The concept: the synthesis of opines by transformed cells creates an ecological niche for the infecting [34] strain of Agrobacterium. 1983 First plant transformed with a recombinant gene using Agrobacterium tumefaciens as a vector. [77] 1984 T-DNA oncogenes are identified that mediate overproduction of and . [26,27] 1985 The virA/virG two-component regulatory system is identified as a central component of signal perception and [20] in Agrobacterium transformation. 1986– Further elucidation of the vir-gene-encoded T-DNA transfer process; identification of plant genes involved in [16–18,47,78,79] present Agrobacterium tumefaciens transformation; extension of A. tumefaciens host range for transformation of monocots; sequencing of the A. tumefaciens (C58) genome. both bacterial [15,16] and plant [17,18] perspectives have genomethroughnon-homologousrecombinationinaprocess recently been published. Briefly, amino acids, organic acids that appears to require plant-encoded proteins (probably and released from wounded plant cells act as enzymes related to DNA repair or recombination) [22,23]. chemoattractants to tumorigenic agrobacteria, which bind to plant cells in a polar orientation upon reaching the Forming a gall in planta wound site [17,19]. Weak attachment to the plant cell is Genes present in the Agrobacterium T-DNA possess the cis first achieved through synthesis of acetylated polysacchar- motifs (e.g. TATA box, CAAT box, polyadenylation signal) ides, followed by strong binding through the extrusion of required for expression in the eukaryotic plant host [24]. cellulose fibrils [17]. Simultaneously, the vir regulon, a set There are two general classes of genes in T-DNA: of required for the transfer of virulent DNA, is oncogenes and opine-related genes. The oncogenes alter activated by the VirA/VirG two-component regulatory phytohormone synthesis and sensitivity in the infected system [20]. The presence of acidic extracellular conditions cell, thus generating the tumor phenotype. T- from (pH 5.0–5.5), phenolic compounds and monosaccharides -type A. tumefaciens strains (e.g. 15955 and Ach5) at a plant wound site directly or indirectly induce contain five oncogenes: iaaM, iaaH, ipt, 6b and 5 [25]. The autophosphorylation of the transmembrane receptor tryptophan mono-oxygenase iaaM converts tryptophan kinase VirA [19]. Activated VirA transfers its phosphate into indole-3-acetamide, and the indoleacetamide hydro- to the cytoplasmic VirG protein, which then binds to the vir lase iaaH catalyzes the synthesis of the box enhancer elements in the promoters of the virA, virB, indole acetic acid (IAA) from indole-3-acetamide (Fig. 1) virC, virD, virE and virG operons, upregulating transcrip- [26]. The ipt gene product mediates the condensation of tion [15]. Through the co-operative action of the VirD1 and adenosine monophosphate with isopentenyl pyrophos- VirD2 proteins, a single-stranded DNA fragment (the phate (iPePP) and/or an unknown terpenoid, forming T-strand) is synthesized from one or more regions of the isopentenyl adenosine 50 monophosphate (iPMP) or zeatin tumor-inducing (Ti) plasmid delimited by specific 25 riboside-50-monophosphate (ZMP) (Fig. 1) [27,28]. This is nucleotide repeat sequences [19]. VirD2 remains cova- the rate-limiting step in cytokinin biosynthesis, and iPMP/ lently bound to the 50 end of the T-strand, which is ZMP are rapidly converted to trans-zeatin by plant- subsequently coated by the VirE2 single-stranded DNA encoded enzymes. The massive accumulation of auxin binding protein, although it is unclear whether VirE2 and cytokinin brought about by the activities of the iaaM, associates with the T-strand in the bacterial cell or in iaaH and ipt enzymes is the primary driver of tumorigen- planta [21]. esis. The secondary oncogenes 6b (tml) and 5 are thought The T-strand/Vir protein complex (T-complex) is primarily to modify the effects of phytohormones in the exported from Agrobacterium to the plant cell cytoplasm cell. The 6b gene product is thought to alter hormone through a type IV bacterial secretion apparatus encoded responsiveness, potentially by increasing sensitivity to by the virB and virD4 [21]. Both VirE2 and VirD2 and decreasing sensitivity to [29,30]. possess nuclear localization sequences and interact with The biochemical nature of 6b activity has yet to be endogenous plant proteins thought to facilitate targeting determined. The product of gene 5 of the T-DNA converts of the T-complex to the nucleus, including an importin-a,a tryptophan into indole-3-lactate, which might act as an type 2C protein phosphatase and three cyclophilins auxin antagonist by competing with IAA for auxin binding (VirD2-interacting factors), and VIP1 and VIP2 (VirE2- proteins [31,32]. interacting factors) [17,18]. The Agrobacterium trans- The second class of T-DNA genes are involved in the ferred DNA (T-DNA) can then integrate into the plant cell synthesis of low molecular weight and http://plants.trends.com 382 Review TRENDS in Plant Science Vol.8 No.8 August 2003 phosphate derivatives called opines. More than 20 have nearly 40 Ti-plasmid encoded genes related to different opines have been identified in crown galls and octopine, agropine and mannopine uptake and use [25]. hairy roots, but only a small subset of these are encoded by Chemically, opines are generally condensation products the T-DNA of any one Agrobacterium strain [33]. Agro- of amino acids, keto acids and sugars, and up to 7% of bacterium strains possess Ti plasmid encoded opine the dry weight of tumor tissue can be composed of uptake and catabolism genes corresponding to the opine [33]. Thus, although not directly involved in particular opine species whose synthesis is directed by tumorigenesis, opines provide a growth substrate for their resident T-DNA. Thus, opine production in trans- Agrobacterium as well as encouraging conjugal Ti formed plant cells creates a distinct ecological niche for the plasmid exchange and chemotaxis [35]. infecting strain of Agrobacterium [34]. The type of opine(s) produced in infected tissues has traditionally been used to classify the infecting strain of Agrobacterium (e.g. octo- Current mechanisms of crown gall disease control pine, and agropine-type strains), although these As with any plant disease, crown gall is a function of the classifications are not always fully inclusive or mutually environment, the pathogen and the plant host [12]. The exclusive. Octopine-type T-DNAs possess four opine absence of a favorable condition for any one of these synthesis genes catalysing the production of octopine elements precludes disease development, and various (ocs), agropine (ags) and mannopine (mas10, mas20) crown gall disease control measures have targeted each [25]. Correspondingly, octopine Agrobacterium strains corner of this ‘disease triangle’ (Fig. 2).

(a) (b) NH2 NH2 OH N H2CCHC N Trp AMP O O N N N

H -O P O CH2 O iaaM O- H H NH2 H H H C C OH OH IAM 2 + + O N O O H CH3 iaaH -O P O P O C C C Unknown terpenoid H2 H CH OH O- O- 3 IAA H C C 2 iPePP ipt ipt O N CH H2C OH H 3 HN C C C HN C C C H H CH3 H2 H CH3 2 N N N N

O N O N N N -O P O CH -O P O CH2 2 O O ZMP O- H H iPMP O- H H H H H H OH OH OH OH

(Reactions catalyzed by plant enzymes)

H2C OH HN C C C H2 H CH3 N N Zeatin N N H TRENDS in Plant Science

Fig. 1. Agrobacterium tumefaciens derived phytohormone biosynthesis pathways. (a) Auxin biosynthesis catalyzed by the iaaM and iaaH oncogenes. (b) Cytokinin biosyn- thesis catalyzed by the ipt oncogene. Adapted from [28,80]. http://plants.trends.com Review TRENDS in Plant Science Vol.8 No.8 August 2003 383

pathogen. Agrobacterium radiobacter strain K84 and its Existing resistant germplasm plasmid-transfer-deficient derivative K1026 are the most Silencing plant genes required for transformation Oncogene silencing widely used and best studied crown gall biocontrol agents, VirE2-mediated resistance although several other Agrobacterium strains have been exploited for control of crown gall in grape [4]. Strain K84 possesses the 48 kb plasmid pAgK84, which encodes Host production of and immunity to the antibiotic agrocin 84 [39]. Agrocin 84 has potent bactericidal activity against A. tumefaciens strains harboring a nopaline-type Ti plasmid [40]. K84 also produces agrocin 434 and ALS 84, additional antibiotic compounds that probably expand the effective range of control beyond nopaline-type A. tumefaciens Crown gall strains [39,41]. Still, pathogenic A. tumefaciens strains disease that are resistant to K84 biocontrol are not uncommon, so crown gall disease control by K84 is not universally effective [42–44]. The bactericidal treatments described Pathogen Environment above are essentially topical, so alternative measures to control Agrobacterium are often required in grape, which is commonly infected systemically by A. vitis. In this case, Biocontrol: Agrobacterium radiobacter K84 Wound prevention A. vitis free propagation material can be produced either Chemical bactericides Phytosanitation by hot water treatment of dormant cuttings or through in Use of Agrobacterium-free propagating material vitro shoot tip culture [36]. TRENDS in Plant Science Plant host Fig. 2. A crown gall disease triangle. Permissive host, pathogen and environmental Although A. tumefaciens has perhaps the broadest host conditions are required for crown gall disease to develop. Various disease control strategies target specific corners of the disease triangle – generating resistance in range of any plant pathogenic bacterium, the agricultural the plant host, eliminating virulent Agrobacterium tumefaciens or preventing impact of crown gall disease is limited to a relatively small environmental conditions conducive to infection. subset of horticultural crops [45,46]. Many cultivated Environment monocots and legumes are not hosts for A. tumefaciens, The primary controllable environmental requirement for although some of these recalcitrant plants (e.g. and the development of crown gall disease is a plant wound. rice) can be transformed by Agrobacterium vectors under Careful cultural practices that prevent unnecessary plant controlled laboratory conditions [47]. The molecular bases of non-host resistance to A. tumefaciens are unknown, wounding can significantly reduce crown gall by denying although the production of antimicrobial metabolites [48], A. tumefaciens an opportunity to introduce T-DNA into a lack of vir gene inducers [49], inefficient T-DNA plant cells [12]. In addition, protection from subfreezing integration into the plant genome [50] and Agrobacter- winter temperatures and control of chewing insects and ium-induced programmed cell death [51] have been nematodes can be crucial in preventing natural wounds proposed as potential mechanisms. that can act as sites of infection [4,12,36]. The soil Among highly susceptible agricultural plant species, a environment can also play a major role in determining great deal of effort has been focused on the identification the incidence and severity crown gall disease. Smith et al. and selection of crown-gall-resistant individuals or culti- [6] prescribed the abandonment of highly infested soils, vars. Varying levels of disease susceptibility have been and intercropping with a non-susceptible host or soil described in plum [52], peach [53], grape [54], aspen [55], fumigation can temporarily reduce soil populations of rose [56] and others. Again, mechanisms of this genotypic Agrobacterium [12,37]. The timely removal of infected and cultivar-level resistance are generally poorly under- plant material can also prevent the continued ‘seeding’ of stood. In the grapevine rootstock ‘Glorie de Montpellier’, soil with large populations of pathogenic A. tumefaciens resistance is manifested during or after T-DNA transfer, derived from crown gall tissues. because A. tumefaciens proliferation, attachment and vir gene induction is comparable to susceptible varieties [54]. Pathogen Crown gall resistance in aspen is negatively correlated Treatments designed to eliminate Agrobacterium directly with cytokinin sensitivity, suggesting that T-DNA- must necessarily be exercised before infection, because mediated phytohormone synthesis is insufficient to disease development will progress independent of the initiate tumorigenesis in resistant cultivars [55]. causal agent following the initial transformation event Nam et al. [57,58] identified several ecotypes and [38]. In situations in which wounding is inevitable, such as T-DNA-tagged mutants of Arabidopsis that are resistant grafting and transplanting, copper- or bleach-based to Agrobacterium transformation (RAT). In some RAT bactericides can reduce A. tumefaciens populations on genotypes, resistance could be attributed to either reduced plant surfaces, minimizing disease [4]. However, biocon- bacterial attachment or inefficient T-DNA integration trol treatments using avirulent Agrobacterium strains [57,58]. T-DNA integration deficiency in the RAT5 mutant that act as A. tumefaciens antagonists have proved to be was found to result from the inactivation of one copy of the the most effective means of controlling the crown gall histone H2A-1 gene [59]. The inheritance of crown gall http://plants.trends.com 384 Review TRENDS in Plant Science Vol.8 No.8 August 2003 resistance is highly variable because dominant, semi- (e.g. vitronectin-like proteins) are probably required for dominant and recessive resistance traits have been bacterial attachment, nuclear import machinery identified among the different Arabidopsis RAT genotypes (e.g. importin-a and VIP1) is required for T-DNA subcellular and among various agricultural species [57,58]. This again trafficking and components of DNA repair and recombina- underlies the multitude of endogenous plant factors tion pathways (potentially including histone H2A-1) appear recruited by Agrobacterium during pathogenesis. to be required for T-DNA integration [17,18]. Post- Inducible plant defenses such as the hypersensitive transcriptional gene silencing (PTGS) of any of these response (HR) and the oxidative burst are commonly plant genes could generate disease resistance by blocking mediated by ‘gene for gene’ interactions between plant the process of Agrobacterium transformation. Indeed, resistance proteins and corresponding pathogen aviru- individual transgenic Arabidopsis plants expressing lence proteins [60,61]. Considering the diversity of crown VIP1 antisense RNA, importin a1 antisense RNA or gall disease resistance mechanisms, it is surprising that histone H2A-1 antisense RNA display crown gall disease relatively few examples of induced resistance have been resistance [64,65] (S. Gelvin, pers. commun.). It must be described in A. tumefaciens–plant interactions [57]. Co- realized, however, that molecular mechanisms exploited inoculation experiments with A. tumefaciens and by Agrobacterium certainly serve other physiological pv. phaseolica have shown that functions in the plant, so silencing of these endogenous Agrobacterium can suppress induction of the HR in plants genes could have undesirable pleiotropic effects. [62]. This HR inhibition was shown to depend on Another PTGS-mediated resistance strategy, oncogene A. tumefaciens auxin synthesis [62]. In addition, silencing, interferes with crown gall disease development A. tumefaciens can detoxify hydrogen peroxide, a primary following T-DNA integration into the plant genome. As component of the plant oxidative burst that has both direct described previously, expression of the T-DNA-encoded germicidal activity and a signaling function in induced oncogenes iaaM, iaaH and ipt in transformed plant cells plant defense [63]. The A. tumefaciens catalase KatA, causes rapid auxin and cytokinin synthesis, which which converts hydrogen peroxide into oxygen and water, initiates and maintains tumorigenesis. These oncogenes is required for virulence on kalanchoe [63]. Thus, it share high nucleotide sequence conservation (,90%) appears that Agrobacterium has developed several unique across all characterized A. tumefaciens strains [66]. strategies to overcome induced plant defense mechanisms. Arabidopsis, tomato (Lycopersicon esculentum) and wal- For many perennial crops, such as walnut and apple, nut (Juglans regia) plants transformed with self-comp- available germplasm resources have not been satisfactory lementary RNA constructs designed to initiate PTGS of sources of crown gall disease resistance. Recently, several iaaM and ipt demonstrated broad-spectrum crown gall biotechnology strategies have been developed for the de resistance (Fig. 3) [66–68]. Resistance was correlated with novo generation of crown gall resistance in plants. These a substantial decrease in iaaM and ipt mRNA in planta strategies generally interfere with the process of and an accumulation of iaaM and ipt-homologous small A. tumefaciens infection or disease development in planta interfering RNAs [66,67]. Although oncogene-silenced without directly targeting the pathogen. plants display normal appearance and development, it As discussed above, it is apparent that a suite of host remains to be seen whether any long-term developmental plant genes is required for efficient Agrobacterium T-DNA penalty is associated with constitutive activity of the PTGS transfer and integration [17,18]. Specific cell wall proteins pathway.

(a) (b)

Fig. 3. Resistance to crown gall mediated by silencing of the T-DNA encoded iaaM and ipt oncogenes. Stems of tomato seedlings were inoculated with Agrobacterium tumefaciens and assayed for disease development five weeks after inoculation. (a) Wild-type plant displaying characteristic massive gall development and stunted growth. (b) An oncogene-silenced transgenic plant displaying normal growth and no gall development (two inoculation sites are visible on the central stem, indicated by arrows). Reproduced, with permission, from [81]. http://plants.trends.com Review TRENDS in Plant Science Vol.8 No.8 August 2003 385

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Five plant scientists chosen as new members by the National Academy of Sciences

Trends in Plant Science congratulates the following plant scientists who were recently elected as members of the National Academy of Sciences in recognition of their outstanding achievements in original research: Anthony R. Cashmore, University of Pennsylvania, Philadelphia, PA, USA Luis Herrera-Estrella, National Polytechnic Institute, Guanajuato, Mexico June B. Nasrallah, Cornell University, Ithaca, NY, USA Michael F. Thomashow, Michigan State University, East Lansing, MI, USA James L. Van Etten, University of Nebraska, Lincoln, NE, USA

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